The long-term goal of this proposal is to delineate how energy utilization in bone cells is regulated and in turn, how substrate availability affects osteoblast differentiation and bone formation. Optimal bone formation is required for peak bone acquisition and skeletal remodeling across the lifespan. We previously showed IGF-I is necessary for terminal OB differentiation through the mTORC1 pathway. Moreover, PTH induces skeletal IGF-I and this peptide is required for the anabolic actions of PTH on bone. Previously we noted that in C57BL/6J (B6) and C3H/HeJ (C3H) mice, the latter exhibits higher bone density, greater bone formation, and more skeletal Igf1 expression. Importantly, C3H COBs have higher rates of oxidative phosphorylation (OxPhos) and glycolysis (Glyc) than B6. We then established that during terminal OB differentiation glycolysis (Glyc) is favored over OxPhos in both strains and that PTH, which stimulates IGF-I, increases Glyc in both COBs and calvariae ex vivo. To understand the role of the IGF pathway in the bioenergetics of OB differentiation, we have been studying the sml/sml mouse that has a defect in mTOR signaling due to a 'loss of function' mutation in the Irs1 gene. These mice have very low bone mass, reduced bone formation but normal OB number and osteoid volume. Remarkably, sml/sml COBs have significantly reduced OxPhos and Glyc during OB differentiation in vitro and in vivo. Taken together these data point to the importance of energy metabolism during bone formation, and the central role of IGF-I in cellular respiration. As such, in this proposal our over- arching hypothesis is that PTH stimulates bone formation by enhancing Glyc through the induction of IGF-I, which in turn activates IRS1/ mTorc/AKT signaling. Thus we propose 2 specific aims in a high risk, high impact strategy to delineate the importance of substrate availability during PTH induced bone formation using novel single cell imaging and bioenergetic studies of OBs and calvariae: 1-Determine the context-specific nature of OB respiration and Glyc as well as its relationship to bone formation in response to PTH in +/+ and sml/sml mice in vitro and ex vivo: We propose there is a time dependent switch during COB differentiation under the influence of PTH when Glyc is activated, leading to enhanced synthesis and mineralization of matrix. Novel in situ imaging and cellular respiration studies will be employed to determine PTH actions on single cell bioenergetics and will be compared with in vitro studies. 2-Define the importance of the IRS/ mTORC1 mTORC2/AKT signaling pathway for PTH-stimulated changes in energy balance in differentiated OBs. We postulate that mTORC2 activation via the IRS/AKT pathway is critical for up regulating Glyc during the OB differentiation following PTH. We will delineate te relationship of individual signaling components of the mTOR pathway to OB bioenergetics. Findings from these studies could lead to novel insights into the fundamental biology of OBs and enhance the possibility of newer anabolic approaches for skeletal disorders.